Bilayer Composition Research Articles

Investigating the Role of Bilayer Size and Composition on Membrane Fluctuations using Large Coarse-Grained Simulations

Local curvature of membranes is suggested to play a role in the regulation of cell signalling pathways. Previous computational studies of membrane fluctuations were restricted to simulating the collective behaviour of ∼1,000 lipids for tens of nanoseconds [1]. With the advent of coarse-grained forcefields, such as MARTINI and the continued advance of computational power we are now able to simulate complex lipid mixtures containing ∼50,000 lipids for several microseconds. Here, we shall examine how the fluctuation modes exhibited by several different ternary lipid mixtures changes as both the size of the bilayer is increased and the amount of cholesterol is altered. Spectral decomposition of the observed fluctuations suggests that large simulations will be essential for dissecting the role of membrane dynamics in the clustering of cell signalling proteins, such as Ras. 1. Lindahl, E., & Edholm, O. (2000). Mesoscopic undulations and thickness fluctuations in lipid bilayers from molecular dynamics simulations. Biophys J, 79:426

How Does Ethanol Affect the Stability of Simple Model Yeast Membranes?

Biofuels such as biomass-based ethanol (EtOH) have become increasingly popular as a source of renewable energy, but the higher cost of production and lower energy content make the fuel difficult to compete with petroleum gasoline. Cellulosic biomass can be used to produce bioethanol, but process efficiency is limited by ethanol's cytotoxic nature. One proposed mechanism for EtOH inhibition of yield growth suggests that ethanol diffuses through cell membranes, decreasing the lipid bilayer stability. Bilayers comprising various molar concentrations of 1-palmitoyl-2-oleoylphosphatidlyserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids were simulated using molecular dynamics. Pure POPS, pure POPE, pure POPC, and mixed 30/70 POPS/POPE lipid bilayers were constructed using the CHARMM36 lipid force field. Each lipid bilayer was exposed to molar concentrations of 0.5%, 1%, 2%, 3%, and 4% ethanol EtOH with additional molar concentrations of 5% and 10% EtOH for pure bilayers only. Average bilayer surface area, NMR deuterium order parameters (SCD), and electron density profiles (EDP) were analyzed to interpret bilayer stability. Each analysis indicated decreased membrane stability with increased EtOH concentration, supporting the proposed mechanism. Trends between different lipid bilayer compositions were all similar. Surface area increases resulted from more frequent EtOH bilayer penetrations into the hydrophobic core, causing bilateral expansion of the membrane's walls. SCD values decreased as more EtOH penetrated the bilayer due to the disruption of lipid packing, increasing membrane fluidity and decreasing stabilizing interactions between hydrophobic lipid tails. As overall EtOH concentration increased, EDP plots indicated increased EtOH concentration within the bilayer, past the hydrophilic head region. EDP plots also indicated decreased bilayer thickness. Increased concentration of EtOH within the bilayer is in accordance with SCD results and decreased bilayer thickness supports increased bilayer surface area results.

Binding of Halictine Antimicrobial Peptides to Model Membranes Composed of POPC:POPG Phospholipids

Halictine antimicrobial peptides were isolated from the venom of the bee Halictus sexcinctus. As described in previous studies, halictinemolecules exhibit antimicrobial action against Gram-positive and -negative bacterial cells. However, they have also a considerable hemolytic activity (LC50 ∼80 µM). Halictine 1 (HAL-1) has 12 amino acid residues (GMWSKILGHLIR-NH2), and +3 charges. Other similar sequence, halictine 2 (HAL-2), has also 12 amino acid residues (GKWMSLLKHILK-NH2), and +4 charges. The interaction of these peptides with model membranes composed of POPC:POPG (3:1, and 1:1 molar ratio) phospholipids were monitored using ITC and optical microscopy. For both bilayer composition, halictines were able to bind to the vesicles, resulting mainly in an exothermic process detected by ITC (ΔHpep = −6.8 kcal/mol, and ΔHpep = −4.5 kcal/mol for HAL-1; ΔHpep = −3.4 kcal/mol, and ΔHpep = −5.1 kcal/mol for HAL-2, for titrations with 3:1, and 1:1 POPC:POPG LUVs respectively). HAL-1 displayed endothermic peaks larger than HAL-2, with an additional heat signal at the end of the binding process that remains unclear. Giant unilamellar vesicles (GUVs) were monitored by optical microscopy, which allowed us to observe a peptide-induced permeabilization of these GUVs, followed by vesicle bursts, which was observed for membranes composed of 3:1, and 1:1 POPC:POPG. It could be an indirect evidence of pore formation, culminating in the total collapse of the GUVs. ITC and microscopy experiments were done with low peptide concentrations (30 µM and 20 µM, respectively), below the lethal dose (LC50) of halictines, estimated as hemolytic. The results suggest that HAL-1 and HAL-2, besides the short amino acid sequences, are efficient to bind to model membranes and, thus, to promote pore formation even with low POPG ratio and also low peptide concentration.

A Systematic Investigation to Determine the Optimal Lipid Coating for Nanopore-Based Sensing Experiments

Despite the importance of proteins, nanopore sensing has so far been mostly focused on single molecule DNA and RNA characterization. One factor that limited experiments with proteins were nonspecific interactions of proteins with the walls of synthetic nanopores. We showed recently, that nanopores with fluid coatings of phospholipid bilayers circumvented this problem. In addition, anchoring proteins to lipid anchors slowed down protein translocation through nanopores and enabled determination of parameters such as the shape, volume, and dipole moment of individual non-spherical proteins. To slow down the translocation time of lipid-anchored proteins further, we have recently formed highly viscous coatings from archaea-inspired lipids. These synthetic lipids are composed of two hydrophilic head groups connected by a long hydrophobic chain; each head group is also attached to an acyl chain that spans half of the membrane thickness. As these lipids contain -in one molecule- the components of a typical lipid bilayer, a single layer is sufficient to form a stable membrane. Experimentation with many lipid coatings, including a variety of archaea-inspired lipids, has revealed differences in translocation of protein, bilayer stability and event frequency. Every bilayer composition tested has been designed to optimize each of these characteristics.

Membrane-Lipid Mediated Rhodopsin Signaling Involves an Ensemble of Conformational Substates

G-protein-coupled receptors (GPCRs) constitute about 50% of the known drug targets. Using rhodopsin as a model GPCR, we study the role of membrane lipids in the rhodopsin activation. Absorption of light by rhodopsin leads to a series of conformational changes that establishes an equilibrium between inactive Meta-I and an ensemble of activated Meta-II states [1,2]. Using UV-visible and FTIR spectroscopy, we show that the distribution of late conformational substates during rhodopsin activation is lipid-mediated as explained by an ensemble activation mechanism (EAM). Lipid bilayer composition (head groups and acyl chain lengths) and membrane protein-lipid interaction govern the EAM through biasing conformational substates. Rhodopsin reconstituted in DOPC backshifts the equilibrium to the Meta-IIa substate, whereas mixed-chain POPC membranes favor the inactive Meta-I state [3]. The wavenumber-dependent analysis of the FTIR-difference spectra yields a distribution of pKa and alkaline endpoint values, consistent with an ensemble of substates for each lipid bilayer-rhodopsin system. A phenomenological Henderson-Hasselbalch function was fitted to the pH titration curves to derive thermodynamic parameters from the spectral analysis. Our thermodynamic studies show that activation of rhodopsin is accompanied by an entropy gain compensating the unfavorable enthalpy increase, analogous to protein unfolding reactions. The results from the EAM analysis are in agreement with the flexible surface model (FSM), which describes elastic coupling of the membrane lipids to integral membrane proteins through a balance of curvature and hydrophobic forces in lipid-protein interactions [4]. Our study also provides insight into the thermodynamic parameters that govern rhodopsin-like GPCR activation in native membrane lipid environments. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A.V. Struts et al. (2014) Meth. Mol. Biol. (in press). [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [4] M.F. Brown (2012) Biochemistry 51, 9782-9795.

The Conformational Dynamics of a Secondary Multidrug Transporter are Modulated by the Lipid Bilayer Composition

LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, acts as a secondary antiporter, catalysing the extrusion of a spectrum of hydrophobic drugs by dissipating a proton gradient. It has evolved to bind and export structurally diverse cytotoxic substances from within the membrane, in contrast to the majority of MFS transporters specialized in the transport of a single soluble substrate, captured from the aqueous medium. Its transport mechanism could thus differ from the alternating access model of the lactose permease, the actual paradigm for all MFS transporters. Recently, we have shown that the protonation of conserved acidic residues is the driving force of the conformational transition, and that substrate binding initiates the cycle by catalysing proton passage (Masureel, Martens et al. 2014). While this analysis was performed on detergent solubilized protein, we now aim to investigate the role of the lipid bilayer in the conformational cycle. For this purpose, we performed Double Electron Electron Resonance (DEER) distance measurements on a library of labeled double-cysteine mutants monitoring both sides of the transporter. Spin labeled mutants were reconstituted in nanodiscs and the distance changes monitored upon changes in the pH or addition of substrate(s). We found that the pH-dependent equilibrium between the inside-open and outside-open conformations is strongly shifted in nanodiscs compared to detergent, suggesting that the pKa of the acidic residues is modified by the lipids. By combining specific lipidic compositions and mutagenesis, we are investigating whether such change in the dynamics are the results of direct phospholipid-protein interaction. Specific structural effects of given lipids are observed: small amounts of cardiolipin allow the conformational transition to occur at physiological pH. These findings could shed light on the mechanism of MFS multidrug transporters in general.

Solubilization of binary lipid mixtures by the detergent Triton X-100: the role of cholesterol.

The solubilization of lipid bilayers of different composition and phase by the detergent Triton X-100 (Triton X-100) was investigated using optical and fluorescence microscopy of giant unilamellar vesicles (GUVs) and light scattering of large unilamellar vesicles (LUVs). The compositions explored were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM), in the gel phase, and binary mixtures of these phospholipids with 30 mol % cholesterol (chol), resulting in bilayers in the Ld and liquid-ordered (Lo) phases, respectively. We show that the phospholipid bilayers are completely soluble in TX-100, but optical microscopy reveals that whereas fluid POPC is gradually solubilized by TX-100, gel SM is first shattered in bilayer fragments. Incorporation of TX-100 in the membrane leads to increase in GUV area, which was quantified and expressed as bound detergent-to-lipid molar ratio. The partition of TX-100 in POPC is very high, decreases in POPC/chol, and is negligible in SM/chol. Fluorescence microscopy shows that TX-100 induces Lo/Ld phase separation in previously homogeneous POPC/chol GUVs, and insoluble bilayer fragments/vesicles are detected with optical microscopy and light scattering. Vesicles of SM/chol, in the Lo phase, are virtually insoluble in TX-100. Taken together, our results show that the presence of cholesterol is the origin of membrane resistance to solubilization, which depending on the specific lipid mixture can result in either partially (POPC/chol) or completely (SM/chol) insoluble mixtures.

Stalk formation as a function of lipid composition studied by X-ray reflectivity

We have investigated the structure and interaction of solid-supported multilamellar phospholipid bilayers in view of stalk formation as model systems for membrane fusion. The multi-component bilayers were composed of ternary and quaternary mixtures, containing phosphatidylcholines, phosphatidylethanolamines, sphingomyelin, cholesterol, diacylglycerol, and phosphatidylinositol. Analysis of the obtained electron density profiles and the pressure–distance curves reveals systematic changes in structure and hydration repulsion. The osmotic pressure needed to induce stalk formation at the transition from the fluid lamellar to the rhombohedral phase indicates how membrane fusion properties are modified by bilayer composition.

Laterally structured ripple and square phases with one and two dimensional thickness modulations in a model bilayer system.

Molecular dynamics simulations of bilayers in a surfactant/co-surfactant/water system with explicit solvent molecules show formation of topologically distinct gel phases depending upon the bilayer composition. At low temperatures, the bilayers transform from the tilted gel phase, Lβ', to the one dimensional (1D) rippled, Pβ' phase as the surfactant concentration is increased. More interestingly, we observe a two dimensional (2D) square phase at higher surfactant concentration which, upon heating, transforms to the gel Lβ' phase. The thickness modulations in the 1D rippled and square phases are asymmetric in two surfactant leaflets and the bilayer thickness varies by a factor of ∼2 between maximum and minimum. The 1D ripple consists of a thinner interdigitated region of smaller extent alternating with a thicker non-interdigitated region. The 2D ripple phase is made up of two superimposed square lattices of maximum and minimum thicknesses with molecules of high tilt forming a square lattice translated from the lattice formed with the thickness minima. Using Voronoi diagrams we analyze the intricate interplay between the area-per-head-group, height modulations and chain tilt for the different ripple symmetries. Our simulations indicate that composition plays an important role in controlling the formation of low temperature gel phase symmetries and rippling accommodates the increased area-per-head-group of the surfactant molecules.

PI3 kinase enzymology on fluid lipid bilayers

We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

Expression of indocyanine green-related transporters in hepatocellular carcinoma

BackgroundIndocyanine green (ICG), an organic anion used in liver function tests, is known to accumulate in hepatocellular carcinoma (HCC) tissues after an intravenous injection. Because the intratumoral expression of transporters for chemical agents influences the behaviors of some malignant tumors, we investigated whether the expression of ICG-related transporters influenced the clinicopathologic features of HCC. Materials and methodsICG accumulation patterns were examined using near-infrared spectroscopy and the protein expression of ICG-related transporters was assessed using immunohistochemistry and immunoblotting in 40 resected HCC specimens. We also determined whether the intratumor expression of these transporters affected intratumor lipid composition by imaging mass spectrometry. ResultsImmunoblot analysis revealed that the expression of organic anion transporting polypeptide 1B3 (OATP1B3) and multidrug resistance p-glycoprotein (MDR)-3, as influx and efflux transporters, respectively, were significantly higher in ICG-accumulated HCC (ICG-high HCC) than in ICG-low HCC. ICG was fluorescently observed in the pseudoglands and bile canaliculi abundantly expressing MDR3. An immunohistochemical examination revealed significantly worse disease-free and overall survival rates in patients with MDR3-negative HCC, in which the intratumoral accumulation of some phosphatidylcholine species was observed under imaging mass spectrometry. ConclusionsThe intratumoral expression of MDR3, a key efflux transporter of ICG, affected the prognosis of patients with HCC, presumably by altering the lipid composition of the lipid bilayers.

Do Lipids Show State-dependent Affinity to the Voltage-gated Potassium Channel KvAP?

As all integral membrane proteins, voltage-gated ion channels are embedded in a lipid matrix that regulates their channel behavior either by physicochemical properties or by direct binding. Because manipulation of the lipid composition in cells is difficult, we investigated the influence of different lipids on purified KvAP channels reconstituted in planar lipid bilayers of known composition. Lipids developed two distinct and independent effects on the KvAP channels; lipids interacting with the pore lowered the energy barriers for the final transitions, whereas voltage sensor-bound lipids shifted the midpoint of activation dependent on their electrostatic charge. Above all, the midpoint of activation was determined only by those lipids the channels came in contact with first after purification and can seemingly only be exchanged if the channel resides in the open state. The high affinity of the bound lipids to the binding site has implications not only on our understanding of the gating mechanism but also on the general experimental design of any lipid dependence study.

DNA-cholesterol barges as programmable membrane-exploring agents.

DNA nanotechnology enables the precise construction of nanoscale devices that mimic aspects of natural biomolecular systems yet exhibit robustly programmable behavior. While many important biological processes involve dynamic interactions between components associated with phospholipid membranes, little progress has been made toward creating synthetic mimics of such interfacial systems. We report the assembly and characterization of cholesterol-labeled DNA origami "barges" capable of reversible association with and lateral diffusion on supported lipid bilayers. Using single-particle fluorescence microscopy, we show that these DNA barges rapidly and stably embed in lipid bilayers and exhibit Brownian diffusion in a manner dependent on both cholesterol labeling and bilayer composition. Tracking of individual barges rapidly generates super-resolution maps of the contiguous regions of a membrane. Addition of appropriate command oligonucleotides enables membrane-associated barges to reversibly exchange fluorescent cargo with bulk solution, dissociate from the membrane, or form oligomers within the membrane, opening up new possibilities for programmable membrane-bound molecular devices.

The Many Faces of Lipid Rafts

The Many Faces of Lipid Rafts

Imaging Supported Interfacial Layers – Factors that Influence Interface Organization and Dynamics

Supported interfacial structures are centrally important to a variety of electrochemical and chemical sensing applications. Lipid bilayer structures have received widespread attention not only because of their potential utility in creating biomimetic devices but also because of their inherent complexity and sensitivity to their environment. Understanding the factors that influence organization and dynamics in such systems could be of immediate relevance to the chemical sensing and biological communities. Our specific interest lies in the formation of interfaces that could be used to host transmembrane proteins for sensing applications. Knowledge of the relationships between bilayer composition and morphology, as well as the influence of overlayer constituents, is a necessary first step in the creation of useful supported bilayer interfaces. We have constructed model bilayer structures containing phosphocholine, sphingomyelin and cholesterol supported on mica, and have examined the organization of and dynamics within these phase-segregated structures using time-resolved fluorescence lifetime and anisotropy imaging. Our results show that these supported bilayer structures cannot be explained simply in the context of phase segregation of cholesterol and phosphocholine domains, and that the organization of these bilayers depends sensitively on the constituents present in liquid overlayers. Our image and dynamic data provide a useful starting point for the construction of biomimetic bilayer structures capable of housing biomolecules for sensing applications.

The Mechanism of Membrane Disruption by Cytotoxic Amyloid Oligomers Formed by Prion Protein(106–126) Is Dependent on Bilayer Composition

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106-126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106-126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated.

LysPGS formation in Listeria monocytogenes has broad roles in maintaining membrane integrity beyond antimicrobial peptide resistance

Listeria monocytogenes is an intracellular, foodborne gastrointestinal pathogen that is primarily responsible for causing listeriosis or food poisoning in otherwise healthy individuals. Infections that arise during pregnancy or within immune compromised individuals are much more serious resulting in the risk of fetal termination or fetal fatality postpartum in the former and septicemia or meningitis with a 20% fatality rate in the latter. While the roles of internalin proteins and listeriolysin-O in the infection process are well characterized, the specific roles of lysine-modified phospholipids in the membrane of L. monocytogenes are not. Investigation into the lipid bilayer composition of L. monocytogenes indicated that the overall proportions of lipids, including lysylcardiolipin and lysylphosphatidylglycerol (LysPG), vary with growth temperature and growth phase. In addition, we demonstrate that LysPG formation is essential for L. monocytogenes survival in the presence of increased osmolytic stress but has no effect on bacterial adherence, invasion or survival in the presence of physiologically relevant concentrations of human neutrophil peptide (HNP-1). In the absence of LysPG synthesis, L. monocytogenes unexpectedly retained flagellum-mediated motility at 37 °C. Taken together, these findings show that LysPG formation in L. monocytogenes has broader functions in virulence and survival beyond its known role in the modification of membrane potential previously observed in other bacteria.

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl+) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H+). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H+ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H+ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.